[Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy].

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1ß(IL-1ß) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1ß and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

[Study on protective effect of water extract from Sabia parviflora on liver injury in mice induced by acetaminophen].

The aim of this study was to observe the protective effect of water extract from Sabia parviflora on mice with acute liver injury induced by acetaminophen, and investigate its possible mechanism. Fifty-eight Kunming mice were divided into 6 groups, 8 in the normal group, 10 in the model group, 10 in the biphenyl diester group, and 10 each in the low, medium and high dose groups. After adaptive feeding for one week, the mice in normal group were intragastrically administered with an equal volume of 0.5% sodium carboxymethylcellulose sodium(CMC-Na), and the mice in other groups were intragastrically administered with corresponding drugs at 20 mL·kg~(-1) once a day. Then acetaminophen(200 mg·kg~(-1)) was administered after the above drug administration except the normal group. The behavior and signs of the experimental animals were observed every day and the samples were taken for experiments on the next day of the final administration. The liver mass and mass index were calculated. The blood was collected from the abdominal aorta and centrifuged to obtain the serum for detecting aspartate aminotransferase(AST) activity and alanine aminotransferase(ALT) activity. The liver tissue homogenate was used to detect superoxide dismutase(SOD) activity, glutathione(glutathione, r-glutamyl cysteingl+glycine, GSH) activity and malondialdehyde(MDA) content. Liver tissue was analyzed for histological analysis. The results showed that S. parviflora could alleviate the lipid peroxidation damage in the liver caused by acetaminophen, reduce the ALT and AST activities in serum, increase the levels of SOD and GSH in liver tissue, decrease the content of MDA in liver tissue, and inhibit the apoptosis. S. parviflora could also improve the live histopathological profile, protect liver cells and restore liver function. Among them, the high dose had the most significant effect and showed dose-effect relationship. This study indicated that S. parviflora had a significant protective effect on acetaminophen-induced liver injury in mice, and its mechanism may be related to its anti-oxidation effect and inhi-bitory effect on apoptosis.

[Therapeutic effect of total triterpenoids of Chaenomeles speciosa combined with omeprazole on gastric ulcer induced by indomethacin in rats].

The aim of this paper was to observe the combination therapy with total triterpenoids of Chaenomeles speciosa and omeprazole on indomethacin-induced gastric ulcer in rats, and explore its possible mechanism. Rats were randomly divided into normal group, model group, omeprazole monotherapy(3.6 mg·kg~(-1)) group, total triterpenoids of C. speciosa monotherapy(100 mg·kg~(-1)) group, total triterpenoids of C. speciosa and omeprazole combination therapy(100 mg·kg~(-1)+3.6 mg·kg~(-1)) group. Except for the normal group, the other groups were given indomethacin(20 mg·kg~(-1)) by oral once a day for 7 consecutive days. Then the treated groups were given corresponding drugs by gavage, once a day for 14 consecutive days. The next day after the last administration, half of the rats in each group were measured the gastric mucosal blood flow, gastric juice volume and serum TNF-α, IL-1ß, IL-6, IL-4 and IL-10. After the remaining rats in each group were underwent pyloric ligation 4 hours after the last administration, the gastric endocrine volume, pH value and total acidity of gastric secretion were measured, then histological analysis was performed, MPO activity, cAMP content and histomorphological analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of gastric tissue TNF-α,IL-1ß, IL-6, IL-4, IL-10, VEGFA, A_(2A)R; the protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue were detected by Western blot. The results indicated that total triterpenoids of C. speciosa and omeprazole combination therapy might significantly increase gastric mucosal blood flow, gastric mucus volume, reduce gastric endocrine volume, secretion acidity and mucosal damage, decrease the levels of TNF-α,IL-1ß and IL-6, increase the levels of IL-4 and IL-10 in blood and gastric tissue, inhibit the activity of MPO, increase the content of cAMP in gastric tissue, up-regulate the mRNA expressions of VEGFA, A_(2A)R and protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue, elevate p-PKA/PKA, p-CREB/CREB and p-EFGR/EFGR. Moreover, the combination therapy with total triterpenoids of C. speciosa and omeprazole was more obvious than those of two monotherapies. These aforementioned findings suggested that the combination therapy with total triterpenoids of C. speciosa and omeprazole on indomethacin-induced gastric ulcer have significant therapeutic effect on indomethacin induced gastric ulcer in rats, its mechanism might be related to regulating A_(2A)R/AKT/CREB, A_(2A)R/VEGFA, EGF/EGFR and MUC6/TFF2 signaling pathways, inhibiting pro-inflammatory factors, increasing gastric mucosal blood flow, up-regulating mucosal cell proliferation factors and promoting mucosal protective factors.

[Effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by DSS in mice].

To observe the effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by dextran sulfate sodium (DSS) in mice, C57BL/6 mice were randomly divided into normal group, model group, total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) groups and sulfasalazine (250 mg·kg⁻¹) group. The ulcerative colitis (UC) model was induced by orally administering 2.5% DSS to the experimental mice, and the corresponding drugs were given to each group 3 days before the administration with 2.5% DSS. The normal group and the model group were given the equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage continuously for 10 days, q.d. The general conditions of the mice were observed on a daily basis, and the disease activity index (DAI) score was recorded. On the 10th day after the treatment, mice were put to death, the contents of TNF-α, IL-1ß, IL-6, IFN-γ, IL-4 and IL-10 in the blood were detected, colon length was measured, colon mucosa damage index (CMDI) score was calculated, and MPO activity detection and histomorphology analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of E-cadherin, occluding,MUC2 and TFF3; the protein expressions of SIRT1, IKKß, p-IKKß, IκBα, p-IκBα and cytosol and nucleus PPARγ, NF-κBp65 in intestinal tissue were detected by western blot. The results indicated that total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) could significantly improve the general conditions of UC mice, reduce the DAI, CMDI and histopathological scores, increase the colon length, reduce the colonic mucosa ulcers, erosion and inflammatory infiltration, restore the normal intestinal mucosal barrier function, reduce the contents of TNF-α, IL-1ß, IL-6, IFN-γ, increase the contents of IL-4 and IL-10 in the blood, inhibit MPO activity in colon tissue, up-regulate the mRNA expressions of E-cadherin, occludin, MUC2 and TFF3 in colon tissue, down-regulate the protein expressions of cytosol PPARγ, tissue p-IKKß, p-IκBα and nucleus NF-κBp65 in the colon tissue, decrease the p-IKKß/IKKß and p-IκBα/IκBα ratios, up-regulate the protein expressions of nucleus PPARγ, tissue SIRT1 and cytosol NF-κBp65 (P<0.05 or P<0.01, respectively), with a dose-effect relationship between the total triterpenoids of C. speciosa treated groups. These findings suggested that total triterpenoids of C. speciosa had a significantly therapeutic effect on UC mice induced by DSS, its mechanism might be related to the regulation of PPARγ/SIRT1/NF-κBp65 signaling pathway, the inhibition of pro-inflammatory factor formation and the up-regulation of protein expression of protective factors.

Two new steroidal glycosides with unique structural feature of 14α-hydroxy-5ß-steroids from Reineckia carnea.

Two new steroidal glycosides (1-2) were isolated from the roots of Reineckia carnea, together with three known compounds (3-5). Their structures were determined on the basis of chemical methods and spectral data. Compounds 1-2 were the unique steroidal glycosides possessing structural feature of 14α-hydroxy-5ß-steroids, and compounds 4-5 were isolated from R. carnea for the first time. The isolated compounds (1-5) were tested in vitro for their cytotoxic activities against the A549, HepG 2 and Caski cancer cell lines. Among them, the compounds 2 and 3 showed cytotoxicity against Caski cancer cell line with IC50 values of 34.4 and 3.7µM, respectively.

Nickel Oxide Nanoparticle-Deposited Silica Composite Solid-Phase Extraction for Benzimidazole Residue Analysis in Milk and Eggs by Liquid Chromatography-Mass Spectrometry.

A novel nickel oxide nanoparticle-deposited silica (SiO2@NiO) composite was prepared via liquid-phase deposition (LPD) and then employed as a solid-phase extraction (SPE) sorbent. When the SPE was coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) analysis, an analytical platform for the sensitive determination of benzimidazole residues in egg and milk was established. The limits of detection of nine benzimidazoles were in the range of 0.8-2.2 ng/mL in milk and 0.3-2.1 ng/g in eggs, respectively, which was 5-10 times superior to the methods with other adsorbents for SPE. The recoveries of nine benzimidazoles spiked in milk and egg ranged from 70.8 to 118.7%, with relative standard deviations (RSDs) being less than 18.9%. This work presented the excellent extraction performance of NiO on benzimidazoles for the first time, and the applicability of the LPD technique used as sorbents for trace analysis in complex matrices was also demonstrated.

[Estrogenic Activities of Alcohol Extract from Phellinus lonicerinus].

Objective: To investigate the estrogenic activities of alcohol extract from Phellinus lonicerinus( AEPL). Methods: Estrogen and anti-estrogen effects were evaluated by cell proliferation experiment in vitro. Through elevating young rat uterine weight, castrated female rats, and adult female rats uterus index serum estradiol( E2) and progesterone( P) were analyzed by enzyme immune methods, and uterine estrogen receptor α( ERα) and estrogen receptor ß( ERß) protein expressions were measeured by immunohistochemisty, and investigated the histopathological of uterus, ovary, and breast of adult female rats. Results: Compared with the control group, AEPL promoted estrogen-sensitive MCF-7 proliferation significantly( P < 0. 05 or P < 0. 01) in the doses of 5 ~ 50 µg / m L in vitro experiment; compared with the E2 control group, it also presented anti-estrogenic effect in E2-induced MCF-7 cells at the doses of 10 ~ 100 µg / m L( P < 0. 05 or P < 0. 01). In the animal experiments, AEPL remarkably increased serum E2 content and promoted growth of uterus in primary female mice at the dose of 300 mg / kg; and raised the serum E2 and P content, alleviated uterine atrophy caused by estrogen deficiency in castrated rats at the dose of 240 mg / kg. In adult female rats, AEPL markedly increased the serum P content at the dose of 120 mg / kg, and also markedly increased the serum E2 content at the dose of 120,240 mg / kg, and regulated the protein expressions of ERαand ERß. AEPL has no effects on histopathological changes of uterus, ovary and mammary gland in rats. Conclusion: AEPL shows estrogenic effects with fewer adverse reaction, which possesses the replacement of estrogen application prospects.

[Research on Chemical Constituents and Anti-tumor Activity of Eupatorium chinense].

Objective: To study the chemical constituents and their anti-tumor activity of Eupatorium chinense. Methods: The chemical constituents were separated and purified by the normal phase silica gel column chromatography,preparative thin-layer chromatography,and preparative HPLC. Their structures were determined by various spectral data,their antitumor activity in vitro was determined by MTT assay. Results: Six compounds were isolated from the ethyl acetate extract of Eupatorium chinense,and the structures were identified as eupalinilide G( 1),8ß-( 4'-hydroxytigloyloxy)-5-desoxy-8-desacyleuparotin( 2),3-( hydroxymethyl)-1,13,14,15-tetrahydroxy-7,11,15-trimethyl-2,6,10-hexadecatriene( 3),3-( hydroxymethyl)-1,13,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-14-yl acetate( 4),eupafolin( 5) and hiyodorilactone B( 6). Compound 2 showed cytotoxicity against HGC-27 and B16 cancer cell lines with IC50 values of 4. 29 µg/m L and 5. 53 µg/m L,respectively. Methods: The chemical constituents were separated and purified by the normal phase silica gel column chromatography,preparative thin-layer chromatography,and preparative HPLC. Their structures were determined by various spectral data,their antitumor activity in vitro was determined by MTT assay. Results: Six compounds were isolated from the ethyl acetate extract of Eupatorium chinense,and the structures were identified as eupalinilide G( 1),8ß-( 4'-hydroxytigloyloxy)-5-desoxy-8-desacyleuparotin( 2),3-( hydroxymethyl)-1,13,14,15-tetrahydroxy-7,11,15-trimethyl-2,6,10-hexadecatriene( 3),3-( hydroxymethyl)-1,13,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-14-yl acetate( 4),eupafolin( 5) and hiyodorilactone B( 6). Compound 2 showed cytotoxicity against HGC-27 and B16 cancer cell lines with IC50 values of 4. 29 µg/m L and 5. 53 µg/m L,respectively. Conclusion: Compounds 2 ~ 5 are isolated from the Eupatorium chinense for the first time,and compound 2 has significant cytotoxic activity against HGC-27 cell line.

Synthesis and cytotoxic activities of 2-substituted (25R)-spirostan-1,4,6-triene-3-ones via ring-opening/elimination and 'click' strategy.

To develop more effective antitumor steroidal drugs, we synthesized a library including twenty-two novel cytotoxic 2-alkyloxyl substituted (25R)-spirostan-1,4,6-triene-3-ones and corresponding 1,2,3-triazoles through an abnormal monoepoxide ring-opening/elimination and 'click' reactions. After the cytotoxic evaluations against HepG2, Caski and HeLa cell lines, three steroidal triazoles 5b, 5f and 5m in this library were found to possess potent anti-proliferative effects against Caski cells with the half-inhibitory concentrations (IC50) of 9.4-11.8 µM. The high-efficient and straightforward process was attractive feature for facile preparation of anti-tumor steroidal triazoles.

Protective effect of tomatine against hydrogen peroxide-induced neurotoxicity in neuroblastoma (SH-SY5Y) cells.

OBJECTIVES: Reactive oxygen species can induce cell apoptosis, and oxidative stress has been implicated in a variety of neurodegenerative disorders. Tomatine, which is a naturally occurring steroidal glycoalkaloid isolated from Solanum cathayanum, has shown potent anti-oxidant properties. METHODS: In this study, we used the SH-SY5Y cell line as an in vitro model and investigated the protective effect of tomatine against hydrogen peroxide (H2 O2 )-induced neurotoxicity in SH-SY5Y cells. KEY FINDINGS: Tomatine might inhibit the release of cellular lactate dehydrogenase, increase anti-oxidant enzyme activity and glutathione content, reverse the downregulated protein expression of the brain-derived neurotrophic factor (BDNF), inhibit expression of Bax and activations of caspase-3 and caspase-9 in H2 O2 -induced SH-SY5Y cells. CONCLUSIONS: Tomatine exerted beneficially neuroprotective effect on H2 O2 -induced SH-SY5Y cells, mainly enhancing intracellular anti-oxidant enzyme activity and BDNF expression, inhibiting H2 O2 -induced oxidative stress as well as expression of Bax and activations of caspase-3 and caspase-9, alleviating H2 O2 -induced SH-SY5Y cell injury and cell death.

Antiproliferative activity of the total saponin of Solanum lyratum Thunb in Hela cells by inducing apoptosis.

Total saponin of Solanum lyratum Thunb (TSSLT), a species of natural biologically active substances isolated from Solanum lyratum Thunb, possesses various bioactivities. It has been proposed that the induction of apoptosis may be the basis of its antitumor activity. However, the molecular mechanism underlying the total saponin-induced apoptotic process remains unknown. In the present study, we describe the anti-proliferative effect of TSSLT on human cervical cancer cells (Hela). The TSSLT induced apoptosis of Hela in a time-dependent manner with an IC50 for cell viability of 6 microg/ml. The TSSLT-induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-like activities, poly (ADP-ribose) polymerase (PARP) cleavage and release of cytochrome c (cyt c) into cytosol. TSSLT activated various caspases such as caspase-3, -8, and -9 (like) activities but not caspase-1 like activity. The cell death was completely prevented by the pan caspase inhibitor benzyloxy carbonyl-Val-Ala-Asp- fluoromethyl-ketone (Z-VAD-FMK). More than 80% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, treatment with TSSLT induced the increase of Bax:Bcl-2 ratio in Hela cells. These results suggest that the induction of apoptosis by TSSLT involves multiple pathways antigen including death receptor and mitochondrial pathway and strongly suggest that the mitochondrial pathway was mediated by low expression of Bcl-2 and upregulation of Bax, release of cyt c and subsequent activation of caspase-3 followed by down stream events leading to apoptotic cell death.

Simultaneous determination of oxidized and reduced glutathione in eel's (Monopterus albus) plasma by transient pseudoisotachophoresis coupled with capillary zone electrophoresis.

Both the reduced form of glutathione (GSH) and the oxidized form of glutathione (GSSG) in eel's ( Monopterus albus) plasma were for the first time determined by transient pseudoisotachophoresis coupled with capillary zone electrophoresis. The method of transient pseudoisotachophoresis coupled with capillary zone electrophoresis has been thoroughly optimized and adequately evaluated for the simultaneous determination of GSH and GSSG in eel's plasma. The detection limits (S/N = 3) of the method developed were 0.2 and 0.05 micromol/L for GSH and GSSG, respectively. The linearity of the calibration curves was valid in the range of 0-10 micromol/L GSH and 0-0.70 micromol/L GSSG. The method was simple, fast, and reproducible. It was found that the respective concentrations of GSH and GSSG were in the range of 9.1-14.5 and 0.31-0.58 micromol/L in the adult eel's plasma, and 10.8-17.9 and 0.49 - 0.68 micromol/L in the juvenile eel's plasma of the three populations determined. Each blood sample was a composite of five eels. For each of the three populations, the concentrations of GSH and GSSG in the adult eel's plasma were lower than those in the juvenile eel's plasma, and the concentrations of GSH and GSSG in the plasma of population 1 (deep yellow finless eels) were higher than those in populations 2 (light yellow finless eels) and 3 (green finless eels) for either the adult or the juvenile eels.

[Effect of mechanical stimulation on the expression of E-cadherin/cateninin complex in salivary adenoid cystic carcinoma].

OBJECTIVE: To investigate the effects of cyclic biaxial mechanical stimulation on adhesion of salivary adenoid cystic carcinoma(SACC) extracellular matrix (ECM) and expression of E-cadherin/cateninin complex in the SACC high metastasis cell lines ACC-M, SACC low metastasis cells line ACC-2, we observed the functions of mechanical stimulation in the adhesion of SACC cell-ECM and investigate the mechanism in the adhesion of ACC. METHODS: Mechanical stimulation were applied to the cells for periods of 1, 3 and 6 hours every day, lasting for 2 days. The amplitude of mechanical stimulation applied to the cells were 1000, 4000 micro strain, at a frequency of 3 Hz. Unstrained cells were used as control. The expression of E-cadherin/cateninin complex on the cell of ACC-M, ACC-2 were studied with laser scanning confocal microscope and image analysis. SACC cell-ECM adhesion was assayed by MTT technique. RESULTS: The results showed that expressions of alpha, beta, gamma-cateninin on the cell of ACC-2 were obviously higher than that ACC-M and E-cadherin on the cell of ACC-M were obviously higher than that ACC-2 without mechanical stimulation. Mechanical stimulation can change the expression of E-cadherin/cateninin complex on the cell of ACC-2 and ACC-M with time. The results also showed that cell-ECM adhesion on the cell of ACC-2 were obviously higher than that of ACC-M without mechanical stimulation. Mechanical stimulation can change the cell-ECM adhesion of ACC-2 and ACC-M with time. CONCLUSION: Mechanical stimulation can change the adhesion of the SACC cell-ECM and expression of E-cadherin/cateninin complex of the SACC cell. We think it played an important role in metastasis of the cancer.

CPU0213, a novel endothelin receptor antagonist, ameliorates septic renal lesion by suppressing ET system and NF-kappaB in rats.

AIM: To examine whether a novel endothelin receptor antagonist, CPU0213, is effective in relieving the acute renal failure (ARF) of septic shock by suppressing the activated endothelin-reactive oxygen species (ET-ROS) pathway and nuclear factor kappa B (NF-kappaB). METHODS: The cecum was ligated and punctured in rats under anesthesia. CPU0213 (30 mg .kg(-1).d(-1), bid, sc X 3 d) was administered 8 h after surgical operation. RESULTS: In the untreated septic shock group, the mean arterial pressure and survival rate were markedly decreased (P<0.01), and heart rate, weight index of kidney, serum creatinine and blood urea nitrogen, 24 h urinary protein and creatinine were significantly increased (P<0.01). The levels of ET-1, total NO synthetase (tNOS), indusible nitric oxide synthetase (iNOS), nitric oxide (NO), and ROS in serum and the renal cortex were markedly increased (P<0.01). The upregulation of the mRNA levels of preproET-1, endothelin converting enzyme, ET(A), ET(B), iNOS, and tumor necrosis factor-alpha in the renal cortex was significant (P<0.01). The protein amount of activated NF-kappaB was significantly increased (P<0.01) in comparison with the sham operation group. All of these changes were significantly reversed after CPU0213 administration. CONCLUSION: Upregulation of the ET signaling pathway and NF-kappaB play an important role in the ARF of septic shock. Amelioration of renal lesions was achieved by suppressing the ET(A) and ET(B) receptors in the renal cortex following CPU0213 medication.